This application seeks to identify substrates of cyclin D-dependent kinases with critical roles in carcinogenesis and cell cycle-based cancer therapy. Mutations in the genes that control the cell cycle are among the most common genetic changes in cancer cells. Although many cell cycle proteins are implicated in cancers, the cyclin D-dependent kinases, CDK4 and CDK6, are fundamentally linked to carcinogenesis. Mutations that deregulate CDK4/6 activity are common in tumors, and CDK4/6 activity is critical for tumor cell maintenance. The essential roles of CDK4/6 in tumorigenesis stimulated the development of pharmacologic CDK4/6 inhibitors that may finally realize the promise of cell cycle-based cancer therapy. However, unlike other CDKs, which phosphorylate many substrates, very few CDK4/6 substrates are known. Indeed, almost all previous work has focused on the Retinoblastoma protein family, and the paucity of other known substrates has limited insights into the mechanisms of CDK4/6- associated cancer and therapy targeting these kinases. Despite the need to identify the CDK4/6 substrates relevant for tumorigenesis and therapy, numerous technical challenges have impeded efforts to identify these proteins. We have developed proteomic methods utilizing ATP analog-sensitive (AS) CDKs and substrate thiophosphorylation that enable us to efficiently identify CDK substrates, and have implemented this approach, termed in situ substrate detection, so as to closely mimic the physiologic environment of CDK substrate phosphorylation. We will now apply these methods to identify CDK4/6 substrates in cancers, and our preliminary studies have demonstrated their ability to detect endogenous and novel CDK6 substrates. This application's goal is to apply these innovative proteomic methods to identify novel CDK4/6 substrates that contribute to tumorigenesis, as well as to evaluate the efficacy of CDK4/6 inhibitors. If successful, this research will define new activities of CDK4/6 that may drive carcinogenesis, will enable future mechanistic studies of CDK4/6-associated cancer and pharmacologic CDK4/6 inhibitors, and may lead to new therapeutic targets.